The turnover of proteins in the cell can be controlled by the covalent attachment of polyubiquitin. Many proteins are known to be degraded by this mechanism, but perhaps the most important of these are regulatory proteins such as the tumor suppressor p53, cyclins, and CDK inhibitors. Other proteins, such as the chloride channel of cystic fibrosis, some G- proteins, NF-KB and other transcriptional activators, receptors, kinases, and many oncoproteins, are also known to be degraded by the ubiquitin system. Ubiquitin is known to target proteins for degradation by a 2- Megadalton complex known as the 26S proteasome, but the mechanism of targeting is unknown. This research proposal examines the role in proteolysis of the largest component in the yeast 26S proteasome, Nas1. A highly conserved, nine-fold repeated sequence motif was recognized in Nas1. Based on analogy to other proteins having related repeat segments, this motif implicates Nas1 in protein binding through a hydrophobic surface of parallel beta strands. My objective is to identify proteins that bind to this motif. Special attention will be give to possibility that the repeats binds either the ubiquitin or substrate component of proteolytic substrates. The specific aims are (1) To construct glutamine scanning mutants in the repeat domain of Nas1. (2) To characterize the effect of these Gln-scanning mutations on proteasome assembly and function, and (3) To test whether the free, non-proteasomal form of Nas1 in the 26S proteasome, and the role of the conserved sequence motif is examined in detail, using a combination of genetic and biochemical methods.